ELECTROSTATIC AND PACKING MUTANTS IN STAPH NUCLEASE

我们研究计划的长期目标是更好地了解 蛋白质折叠和结合的原理。 这个根本问题是 与更好地理解广泛的生物系统有关 和问题 我们正从四个方面着手解决这些问题。 所有 这些研究是为大学生工作者设计的。 首先，我们在一个更稳定的环境中， 我们的模型蛋白葡萄球菌核酸酶的变体。 的变化 将测定这些突变蛋白质随pH的稳定性。 该数据 能让我们确定这个地方的微观介电常数 在蛋白质内部。 准确的实验测定 介电常数将增加建模的准确性， 蛋白质中的静电相互作用第二，我们计划建立一个大型的 核酸酶的脂肪族残基的单包装突变体的数量。 我们 会用异亮氨酸和缬氨酸取代所有的亮氨酸， 亮氨酸和缬氨酸，每一个缬氨酸都有亮氨酸和异亮氨酸。 的 将测定这些突变体的热力学稳定性。 该数据 将使我们能够检查包装在稳定这种蛋白质方面的作用 结构，并提供一个基本的数据集，以补充生成的数据， 从我们的第三个方法。 第三种方法是选择两个子集， 在核酸酶的核心中有四个脂肪族残基， 在这四个位点上的异亮氨酸、亮氨酸和缬氨酸的排列。 我们将 也在核心的六个位置取样可能的突变体。 库 在核酸酶核心中有大约210-220种不同的多突变体排列 将由此产生。 蛋白质的热力学稳定性将 被确定。 这个数据集将是最大的一组多个突变体 具有完整的热力学特性。 加上来自 在分子中的每个脂肪位点的单个突变， 一组极好的测试数据，用于模拟蛋白质内部的堆积。 我们最后一项计划是克隆、表达和纯化两个 发现了与葡萄球菌核酸酶同源的蛋白质。 的 这些蛋白质的结构和功能相对于 核酸酶将提供有用的信息，以补充大量的 对核酸酶结构和功能的研究现状进行了综述。

The long range goals of our research program are to better understand the principles of protein folding and association. This fundamental problem is relevant to a better understanding of a wide range of biological systems and problems. We are attacking the problems through four approaches. All these studies are designed to be carried out by undergraduate workers. First, we making six polar mutations at a buried site in a more stable variant of our model protein staphylococcal nuclease. The variation of the stability of these mutant proteins with pH will be determined. This data will allow us to determine the microscopic dielectric constant of this site in the protein interior. An accurate experimental determination of the dielectric constant would increase the accuracy of modeling of electrostatic interactions in proteins. Second, we plan to make a large number of single packing mutants of the aliphatic residues of nuclease. We will substitute every leucine with isoleucine and valine, every isoleucine with leucine and valine, and every valine with leucine and isoleucine. The thermodynamic stability of these mutants will be determined. This data will allow us to examine the effects of packing in stabilizing this protein structure and provide a basic dataset to complement the data generated f rom our third approach. This third approach is to select two subsets of four aliphatic residues in nuclease's core and make all 81 possible mutant arrangement of isoleucine, leucine and valine at these four sites. We will also sample the possible mutants at six sites in the core. A library of some 210-220 different multiple mutant arrangements in the nuclease core will thus be generated. The thermodynamics stability of the proteins will be determined. This dataset will be the largest set of multiple mutants with complete thermodynamic characterization. Together with the data from the single mutations at every aliphatic site in the molecule we will have a splendid set of test data for modeling the packing of protein interiors. Our last initiative will be to clone, express, and purify two recently discovered proteins that are homologous to staphylococcal nuclease. The characterization of the structure and function of these protein relative to nuclease will provide useful information to complement the large number of existing studies of nuclease's structure and function.